Method for rapid identification of pharmacologically active chemical entities associated with the efficacy of ethnobotanical substances

ABSTRACT

The method includes the steps of performing in-vitro liver, intestinal and/or expressed enzyme assays with selected ethnobotanical substances, for both humans and a variety of animal species, to produce an array of resulting chemical entities, such as metabolites, for the human and the animals. Comparisons are then made between the chemical entities from the human in-vitro studies and the animal in-vitro studies to determine the closest match. The animal with the closest match is then used for an in-vivo study. If a match is present between the animal in-vivo results and the human in-vitro results, the matched chemical entity is isolated or synthesized and then further tested to determine the suitability of the matched chemical entity as a treatment drug.

TECHNICAL FIELD

This invention relates generally to methods of drug discovery anddevelopment, and more specifically concerns such a method which issubstantially faster in identifying safe and effective drugs producedfrom ethnobotanical substances than existing methods.

BACKGROUND OF THE INVENTION

In typical modern drug discovery methods, a target biomolecule whichcauses disease is first identified. Typically, the biomolecule is aprotein. The targets are then developed into laboratory-scale assays orscreens. The laboratory-scale screens are converted into automatedscreens for high throughput evaluation of potential drug compounds. Highthroughput screening involves the testing of many different compounds(compound libraries), chosen from a large array of chemicals for theirability to inhibit or otherwise affect the target disease in somespecific desired way. Often, those compound libraries will comprisethousands of chemicals or even more. Those chemicals which lookpromising, as indicated by the results of the high throughput screeningprocess, are then further screened to produce the most promisingleads/candidates. These leads/candidates are first tested with in-vitroassays, and then in-vivo in laboratory animals, to determine if theyproduce activity against the target disease. A compound which passesthis assay testing process will then typically undergo the conventionaldrug discovery and development process involving various testingprocedures and clinical trials.

Such conventional methods involve a substantial amount of time and cost,and often produce commercially nonviable compounds. Hence, it would bedesirable to have a drug discovery method which is simpler, morestraightforward, less expensive and more reliable in identifyingeffective disease fighting compounds.

SUMMARY OF THE INVENTION

Accordingly, one embodiment disclosed herein is a method for identifyingmedicinally active chemical entities in ethnobotanical substances,comprising the steps of: performing an in-vitro assay with anethnobotanical substance using human intestinal and/or liver and/orenzyme expression preparations to produce an array of human chemicalentities; performing an in-vitro assay of said ethnobotanical substanceusing animal intestinal and/or liver and/or enzyme expressionpreparations from at least one selected animal species to produce anarray of animal chemical entities; determining any matches between thehuman chemical entities and the selected animal chemical entities toidentify a matched animal species; performing an in-vivo dosing of theethnobotanical substance with the matched animal species; and performingan analysis of a biological fluid from the matched animal species todetermine any matches between the in-vitro human chemical entities andthe in-vivo matched animal chemical entities.

Another embodiment is a method for identifying medicinally activechemical entities in ethnobotanical substances, comprising the steps of:performing an in-vitro assay with an ethnobotanical substance usinghuman intestinal and/or liver and/or enzyme expression preparations toproduce an array of human chemical entities; performing an in-vivodosing of a selected animal species with said ethnobotanical substance;and performing an analysis of a biological fluid from the selectedanimal species to determine any matches between the in-vitro humanchemical entities and the in-vivo animal chemical entities.

Still another embodiment is a method for identifying medicinally activechemical entities in ethnobotanical substances, comprising the steps of:performing an in-vitro assay with an ethnobotanical substance usinghuman intestinal and/or liver and/or enzyme expression preparations toproduce an array of human chemical entities; performing an in-vivodosing of at least one selected animal species with said ethnobotanicalsubstance; performing an analysis of a biological fluid from theselected animal species to determine a match between the in-vitro humanchemical entities and the in-vivo animal chemical entities; if there isno match, then perform an in-vitro assay of said ethnobotanicalsubstance using intestinal and/or liver and/or enzyme expressionpreparations from the same selected animal species to determine anymatches between the in-vitro human chemical entities and the in-vitroanimal chemical entities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram which sets forth the individual steps in themethod disclosed herein.

FIG. 2 is a block diagram showing alternative embodiments to the methodof FIG. 1.

FIG. 3 is an example of a high performance liquid chromatograph massspectrometer (HPLC-MS) output for a chemical compound.

BEST MODE FOR CARRYING OUT THE INVENTION

The present method makes use of established in-vitro study processes,including for example metabolic processes, as well as other processes,to identify chemical entities, both primary and secondary, which areresponsible for the efficacy of ethnobotanical substances, i.e.medicines. The term “chemical entities” as used herein includes, but isnot limited to, metabolites and other chemical entities produced by bodyaction as well as chemical entities present in the ethnobotanicalsubstances themselves and/or extracts thereof. These in-vitro studyprocesses can include various study designs utilizing enzymaticpreparations, such as human and animal intestinal and/or liverpreparations (microsomes, hepatocytes, liver slices, etc.) as well ashuman and animal enzyme expression preparations, such as lymphoblast andbaculovirus-insect cell expression preparations, to produce an array ofhuman and animal chemical entities.

It is well known that ethnobotanical substances, i.e. naturalsubstances, including extracts thereof, from both land and marine plantsources, such as roots, fruits, seeds, bark and leaves, etc. have beenused throughout human history for successful treatment of variousdiseases and maladies. In the recent past, ethnobotanical data has beencarefully evaluated in an effort to discover new chemical compounds,i.e. active agents, associated with the ethnobotanicals which areresponsible for the medicinal effect observed in naturally occurringethnobotanical substances.

The presumption to this point has been that the active agent resides inthe extracts of the ethnobotanical substances, which can be obtained bytraditional fractionation methods. However, numerous extracts obtainedfrom ethnobotanical substances which have seemingly had the potential ofproducing the same significant medicinal effect as the ethnobotanicalsubstances themselves, have had a high failure rate relative toidentifying the active agents in the ethnobotanical substances, despitesophisticated instrumentation and advanced analytical techniques.

The present method is based on a concept disclosed herein, specificallythat the advantageous medicinal effect of various ethnobotanicalsubstances is more likely due to chemical entities, such as for example,metabolites, which are the product of liver enzyme and/or intestinaloxidation or other bodily function which occurs when the ethnobotanicalsubstance is ingested by the human user, and hence not necessarily fromthe chemical entity present per se in the ethnobotanical extract. Thepresent method is designed to rapidly identify the chemical entitieswhich may be responsible for the efficacy of the ethnobotanicalsubstances. Further, the method may identify a library of novelcompounds for further structure activity relationship evaluation.

In a first step in one embodiment (FIG. 1) of the process, shown inblock 12 thereof, human liver microsomal (HLM) assays, as one example,are used to produce in-vitro profiles, i.e. identification, of resultingchemical entities, such as metabolites, from a selected ethnobotanicalsubstance. The term ethnobotanical substance used herein includesextracts thereof. In addition to the liver microsomal assays, furtherexamples of specific preparations which could be used for this in-vitrostep include intestinal and/or other liver preparations as well as humanenzyme expression preparations, such as lymphoblast andbaculovirus-insect cell expression preparations. A liquid chromatographmass spectrometer (LC-MS) and/or gas chromatograph mass spectrometer(GC-MS) analysis of the samples from the in-vitro assays is thenperformed. An example of an LC-MS display from an in-vitro assay for agiven chemical compound is shown in FIG. 3 for illustration.

The same assays are also performed in-vitro with preparations fromvarious animal species, shown in block 14 in FIG. 1. Animal intestinaland/or liver preparations as well as animal enzyme expressionpreparations could be used as well as the preferred liver microsomes,like that used for the human in-vitro assay. The possible animal speciesinclude, for example, among others, mice, rats, dogs, monkeys, etc.While testing of a variety of animals is preferred, it should beunderstood that animal in-vitro testing could be accomplished with justone animal species. It should also be understood that in the method ofFIG. 1, the above two in-vitro assay steps could be reversed in sequenceand the claims herein interpreted accordingly.

The in-vitro results from the selected animal or animals are thencompared with the in-vitro results from the human in-vitro assays. Thebest matches from one or more animal species are then selected (block16) for in-vivo dosing with the ethnobotanical substance, as shown inblock 18. The various terms match, matches or best match as used hereinand in the claims refer to results having a similar chromatographic ormass spectrometric profile, or equivalent standard. One skilled in theart can identify a match as defined above for the purposes of carryingout the present method by applying such a standard, as it is wellunderstood by those skilled in the art. The meaning of the term “similarprofiles” can include, for instance, similarly positioned peaks in thechromatographic or spectrometric data. Also, the term “similar metabolicprofile” is well understood as a suitable standard and can be used forestablishing a match in appropriate situations in the present method.This match determination of the data can be done by a human, utilizingpre-established standards in accordance with the above considerations,or the data can be compared automatically with the use of a computerprogram utilizing conventional correlation methods to determine whetheror not any match is sufficiently close to proceed with in-vivo testing.A combined manual and automatic determination can also be used.

The matched animal species then undergoes a typical dosing (animalfeeding) study (block 18). The dosing will, for example, comprise thefollowing protocol and feeding schedule. The ethnobotanicalmaterial/substance (e.g. leaves, seeds, roots, fruits, etc.) isadministered to the animal in an oral dosing regimen in a capsule,paste, ground material or extract form, using commercially availableformulation vehicles, such as described in the publication titledDrugs—From Discovery to Approval; Rick Ng; Wiley-Lip. 2004.

Following the in-vivo feeding program, selected biological fluidsamples, such as, for example, blood (whole blood or plasma), urine,feces, bile, etc., are collected and analyzed, using an LC-MS and/orGC-MS or other equivalent analytical techniques to display the presenceof the chemical entities, such as metabolites, present in the selectedbiological fluid. This is followed by a comparison with the resultsobtained from the in-vitro human testing. This is shown at block 20 inFIG. 1. A comparison is then made to again determine matches, using theabove-described chromatographic or mass spectrometric similar profilecomparison or equivalent standard. Again, such a comparison to determinea match for the purposes of this method is within the knowledge of oneskilled in the art, and can be done by a human operator, usingpre-established standards, or it can be done automatically, using amachine with a computer program, or by a combination of manual andautomatic steps.

Those (one or more) chemical entities, such as, for example, but notlimited to, metabolites, which satisfy the matching/comparison criteria,if any, are identified as potential active chemical entities which couldpossibly be responsible for the efficacy of the original ethnobotanicalsubstance occurring in nature which has some known medicinal effect,shown for example as metabolite ID in block 21, although it could beother chemical entities (CE) as well.

At this point, well-known methods are used to either isolate orsynthesize the match-determined chemical entity (which could, forinstance, be a metabolite or it could be another chemical entity). Thisis shown at block 22 in FIG. 1. These are well-known commercial methods,such as described, for example, in Modern Methods of Organic Synthesis:W. Carruthers and lain Coldham; Cambridge University Press, 2004.

Following synthesis/isolation of the match-determined metabolite(s) orother chemical entity, conventional drug development methods areutilized. This includes in-vitro or in-vivo pharmacology studies (blocks24, 26) as well as toxicology studies (block 28). These studies can bedone in sequence or in parallel. Metabolites and perhaps other chemicalentities are likely to satisfy the pharmacology evaluation criteria,since the metabolites and other identified chemical entities typicallywill have known safety and activity profiles. Following the pharmacologyand toxicology studies, human clinical trials will be conducted. Theconduct of human clinical trials is well known. Human clinical trialsare discussed in many texts and publications. One example, forillustration, is A Guide to Clinical Drug Research: A. Cohen & J.Posner; Springer, 2^(nd) Ed. 2000. A regulatory filing (block 32)follows, with subsequent commercial use.

In another embodiment, shown in FIG. 2, human in-vitro assays (block 50)can be compared with the results of in-vivo dosing (block 52) of one ormore animal species and a match, if any, determined (block 54). In thisembodiment, the step of animal in-vitro assays (block 56) is not used.Any match-determined chemical entities can then be synthesized, as shownat block 22. With this embodiment, and the next embodiment, the initialindividual steps of human in-vitro assays and animal in-vivo dosing canbe accomplished in any sequence. The remaining steps in FIG. 2 areidentical to the steps in FIG. 1.

FIG. 2 shows yet another embodiment of the method disclosed herein. Inthis embodiment, like that immediately above, human in-vitro assays(block 50) can be compared with the results of in-vivo dosing (block 52)of one or more animal species. If there is no match, then in-vitroassays are done for the same animal, and the in-vitro animal results arecompared with in-vitro human results (block 56). The in-vitro animalassays of the ethnobotanical substances use intestinal and/or liverand/or enzyme preparations from the same animal. Any match-determinedchemical entities, including for example, but not limited to,metabolites, can then be synthesized, at block 22, as shown in FIG. 1.The remaining steps in FIG. 2 are identical to the steps in FIG. 1.

The advantage to the above-described methods is that they eliminate asubstantial amount of time and effort used in current drugdiscovery/development methods which involve target selection,validation, high throughput screening and medicinal chemistryevaluations. The steps of the above methods of identifying chemicalentities which have a high probability of efficacy are rapid andreliable, involving relatively little time and expense. Uponidentification of the high probability chemical entities, traditionalpharmacology studies and toxicology studies, followed by clinicaltrials, can be utilized.

It is advantageous that those chemical entities which ultimately areidentified and enter into the conventional pharmacological andtoxicology studies have a high probability of success in effectiveness.Further, they also may have a high probability of success in toxicologytesting, since the chemical entities, such as metabolites, resultingfrom the ethnobotanical substances (including extracts thereof) oftenalready have a satisfactory safety profile. Following success withclinical trials, the new drug can then be submitted to regulatoryagencies for marketing authorization and subsequent commercial use.

Although a preferred embodiment of the invention has been disclosed herefor the purposes of illustration, it should be understood that variouschanges, modifications and substitutions may be incorporated in theembodiment without departing from the spirit of the invention, which isdefined by the claims which follow.

1. A method for identifying medicinally active chemical entities inethnobotanical substances, comprising the steps of: performing anin-vitro assay with an ethnobotanical substance using human intestinaland/or liver and/or enzyme expression preparations to produce an arrayof human chemical entities; performing an in-vitro assay of saidethnobotanical substance using animal intestinal and/or liver and/orenzyme expression preparations from at least one selected animal speciesto produce an array of animal chemical entities; determining any matchesbetween the human chemical entities and the selected animal chemicalentities to identify a matched animal species; performing an in-vivodosing of the ethnobotanical substance with the matched animal species;and performing an analysis of a biological fluid from the matched animalspecies to determine any matches between the in-vitro human chemicalentities and the in-vivo matched animal chemical entities.
 2. The methodof claim 1, including the further step of synthesizing or isolatingthose in-vitro human and in-vivo animal chemical entities that match foruse in follow-on studies to confirm the suitability of the matchedchemical entities as a treatment drug.
 3. The method of claim 1, whereinin-vitro testing is performed on a plurality of animal species, withthose species producing the closest match of animal chemical entities tohuman chemical entities being used as the matched animal species for thestep of animal in-vivo dosing.
 4. The method of claim 1, wherein thesteps of determining matches are accomplished using pre-establishedstandards.
 5. The method of claim 4, wherein the steps of determiningmatches are carried out automatically.
 6. The method of claim 4, whereinthe steps of determining matches are carried out manually.
 7. The methodof claim 4, wherein the steps of determining matches are carried outwith a combination of automatic and manual steps.
 8. The method of claim1, wherein the human and animal in-vitro assays are liver microsomalassays.
 9. The method of claim 1, wherein the human and animal in-vitroassays are intestinal microsomal assays.
 10. The method of claim 1,wherein the human and animal in-vitro assays are liver hepatocytesassays.
 11. The method of claim 1, wherein the human and animal in-vitroassays are enzyme expression assays.
 12. The method of claim 1, whereinthe biological fluid is whole blood and/or plasma.
 13. The method ofclaim 1, wherein the chemical entities are metabolites.
 14. The methodof claim 1, wherein the human and animal in-vitro assays are livermicrosomal assays, wherein the chemical entities are metabolites andwherein the biological fluid is whole blood or plasma.
 15. The method ofclaim 2, wherein the follow on studies include pharmacology in-vitrostudies, pharmacology in-vivo studies and toxicology studies todetermine the suitability of the chemical entities.
 16. The method ofclaim 1, wherein the biological fluid testing includes the use of LC-MSand/or GC-MS data.
 17. A method for identifying medicinally activechemical entities in ethnobotanical substances, comprising the steps of:performing an in-vitro assay with an ethnobotanical substance usinghuman intestinal and/or liver and/or enzyme expression preparations toproduce an array of human chemical entities; performing an in-vivodosing of at least one selected animal species with said ethnobotanicalsubstance; performing an analysis of a biological fluid from theselected animal species to determine a match between the in-vitro humanchemical entities and the in-vivo animal chemical entities; performingan in-vitro assay of said ethnobotanical substance using intestinaland/or liver and/or enzyme expression preparations from the selectedanimal species if there is not an in-vitro human/in-vivo animal match;and determining any matches between the in-vitro animal chemicalentities and the in-vitro human chemical entities.
 18. The method ofclaim 17, including the step of synthesizing or isolating those in-vitrohuman and in-vivo animal and in-vitro animal chemical entities whichmatch for use in follow-on studies to confirm the suitability of saidmatched chemical entities as a treatment drug.
 19. The method of claim17, wherein in-vivo dosing is performed on a plurality of animalspecies.
 20. The method of claim 17, wherein the steps of determiningmatches are accomplished using pre-established standards.
 21. The methodof claim 20, wherein the steps of determining matches are carried outautomatically.
 22. The method of claim 20, wherein the steps ofdetermining matches are carried out manually.
 23. The method of claim20, wherein the steps of determining matches are carried out withautomatic and manual steps.
 24. The method of claim 17, wherein thehuman and animal in-vitro assays are liver microsomal assays.
 25. Themethod of claim 17, wherein the human and animal in-vitro assays areintestinal microsomal assays.
 26. The method of claim 17, wherein thehuman and animal in-vitro assays are liver hepatocytes assays.
 27. Themethod of claim 17, wherein the human and animal in-vitro assays areenzyme expression assays.
 28. The method of claim 17, wherein thebiological fluid is whole blood or plasma.
 29. The method of claim 17,wherein the chemical entities are metabolites.
 30. The method of claim17, wherein the human and animal in-vitro assays are liver microsomalassays, wherein the chemical entities are metabolites and wherein thebiological fluid is whole blood or plasma.
 31. The method of claim 18,wherein the follow-on studies include pharmacology in-vitro studies,pharmacology in-vivo studies and toxicology studies to determine thesuitability of the chemical entities.
 32. The method of claim 17,wherein the biological fluid testing includes the use of LC-MS and/orGC-MS data.
 33. A method for identifying medicinally active chemicalentities in ethnobotanical substances, comprising the steps of:performing an in-vitro assay with an ethnobotanical substance usinghuman intestinal and/or liver and/or enzyme expression preparations toproduce an array of human chemical entities; performing an in-vivodosing of a selected animal species with said ethnobotanical substance;and performing an analysis of a biological fluid from the selectedanimal species to determine any matches between the in-vitro humanchemical entities and the in-vivo animal chemical entities.
 34. Themethod of claim 33, including the step of synthesizing or isolatingthose in-vitro human and in-vivo animal chemical entities which matchfor use in follow-on studies to confirm the suitability of said matchedchemical entities as a treatment drug.
 35. The method of claim 33,wherein in-vivo testing is performed on a plurality of animal species.36. The method of claim 33, wherein the steps of determining matches areaccomplished using pre-established standards.
 37. The method of claim36, wherein the steps of determining matches are carried outautomatically.
 38. The method of claim 36, wherein the steps ofdetermining matches are carried out manually.
 39. The method of claim36, wherein the steps of determining matches are carried out with acombination of automatic and manual steps.
 40. The method of claim 33,wherein the human in-vitro assay is a liver microsomal assay, whereinthe chemical entities are metabolites and wherein the biological fluidis whole blood and/or plasma.
 41. The method of claim 33, wherein thefollow-on studies include pharmacology in-vitro studies, pharmacologyin-vivo studies and toxicology studies to determine the suitability ofthe chemical entities as a treatment drug.
 42. The method of claim 33,wherein the biological fluid testing includes the use of LC-MS and/orGC-MS data.